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Department of Obstetrics and Gynecology (J.A.C., Y.-J.J., Z.S., M.S.S.), the Sealy Center for Molecular Science (M.S.S.), and the Department of Surgery (K.L.I., M.R.H.), University of Texas Medical Branch, Galveston, Texas 77555
Address all correspondence and requests for reprints to: Melvyn S. Soloff, Ph.D., Department of Obstetrics and Gynecology, University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555-1062. E-mail: msoloff{at}utmb.edu
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Introduction |
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During the past few years, several reports have indicated that OTRs are found in human breast tumor cells of epithelial origin, as measured by immunological techniques (12, 13, 14). There were no clear indications, however, whether these receptors were functional. To determine whether OTR can be up-regulated in breast tumor cells as in other OT target cell types and to identify the factors involved in this up-regulation, we carried out the present studies on Hs578T cells, a human breast carcinosarcoma cell line (15). We also examined signal pathways associated with OT action in other OT target cell types to determine whether the OTRs were functional. We have found that Hs578T cells contain iodinated OT antagonist (OTA) binding sites that are up-regulated by dexamethasone (DEX) and serum. We also demonstrate that the receptors are functional, as measured by OT-induced increases in intracellular calcium concentrations ([Ca2+]i), phosphorylation of ERK-2 kinase (extracellular signal-regulated protein kinase), and stimulation of PGE2 synthesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P]UTP (3000
Ci/mmol), New England Nuclear Corp. (Boston, MA).
Cell culture conditions
Hs578T cells were obtained from American Type Culture Collection (Manassas, VA) and cultured in DMEM supplemented with
10% FBS and penicillin/streptomycin at 37 C under an atmosphere of 5%
CO2. To down-regulate OTR levels in Hs578T cells, FBS was
replaced with 0.5% BSA for 5 days. Cells remained viable through serum
starvation. FBS, treated with dextran-coated charcoal (DCC-FBS) to
remove steroids, was used at a final concentration of 10% unless
otherwise noted. Serum-deprived cells were treated with DCC-FBS, 1
µM DEX, or both to up-regulate OTR levels.
Determination of OTR binding
Cells grown on culture plates were rinsed with ice-cold PBS and
then collected with a rubber policeman into a 20 mM
NaHCO3-5 mM EDTA solution. The cells were
homogenized and centrifuged at 48,000 x g for 30 min
at 4 C. The pellet was suspended in 50 mM Tris-HCl (pH 7.6)
and centrifuged. The pellet was resuspended in 50 mM
Tris-HCl (pH 7.6), 5 mM MgSO4, 1 mM
EDTA, 1 mg/ml BSA, and 100 µg/ml bacitracin and stored at -70 C. OTA
was monoiodinated as previously described (16). The specific activity
of the iodinated peptide was 2,000 Ci/mmol at the time of preparation.
Crude cell membrane fractions from Hs578T cells were prepared and
assayed for binding activity as described previously (16). Separation
of free from bound peptide was carried out by filtration through
Whatman GF/F microporous glass filters (Clifton, NJ) that
were presoaked in 0.3% (wt/wt) polyethyleneimide in water (16).
Ki values of peptide analogs were calculated according to
the method of Cheng and Prusoff (17). Binding experiments, each
comprised of at least eight points, were repeated at least three
times.
Whole cell assays for OTR binding were performed on serum-starved cells subsequently treated with DCC-FBS, DEX, separately and together, and other agents. Briefly cells were rinsed twice in 2 ml PBS and then incubated in 1 ml PBS containing a saturating (or near-saturating) concentration of [125I]OTA at room temperature for 1 h. Nonspecific binding was determined by adding unlabeled OT (1 µM) in combination with [125I]OTA. Cells were then rinsed three times with 2 ml PBS and solubilized with 0.5 ml 1 N NaOH. Radioactivity in the extracts was determined by scintillation counting. The concentration of cellular DNA was determined in parallel, using the Hoechst dye H 33258 and a Hoefer DyNA Quant fluorometer (Hoefer Scientific, San Francisco, CA) according to the manufacturer’s instructions. Results are expressed as counts per min specifically bound per µg DNA.
RT-PCR
RNA from term human myometrium and Hs578T cells was isolated
(18), and RT-PCR was performed using a Perkin Elmer
GeneAmp RNA PCR kit and RNA PCR Core kit (Norwalk, CT). Total RNA (1
µg) was used for first strand complementary DNA synthesis.
Amplification products were analyzed by electrophoresis in 5%
nondenaturing polyacrylamide gels and were visualized by ethidium
bromide staining. DNA markers (BioMarker Low) were obtained from
Bioventures, Inc. (Murpheesboro, TN). The sequences for the upstream
and downstream primers for the human V1a vasopressin
receptor (GenBank accession no. U19906) were 5'-TGCCACCCGCTCAAGACTC-3
(positions 2460–2478) and 5'-GGTGATGGTAGGGTTTTCC-3' (5197–5215),
respectively. These primers are located in the second intracellular and
third extracellular loops, respectively, and are interrupted by a
2.2-kb intron in genomic DNA. The human (h) OTR (GenBank accession no.
X64878) primer sequences 5'-CCTTCATCGTGTGCTGGACG-3' (1215–1234) and
5'-CTAGGAGCAGAGCACTTATG-3' (1586–1605) are located in the sixth
transmembrane region and 3'-noncoding sequence, respectively. The
priming sites in genomic DNA span a 13.2-kb intron (19). The sequences
for the upstream and downstream primers, respectively, for
ß-polymerase were 5'-AGTCCTGGTACCTCCTTCAAGCTG-3' and
5'-GGGTATTTTGCTATAACAGATGCTGCTTTT-3'. The primers were designed to
anneal to sites separated by intron(s) to prevent contamination by
genomic DNA. Correct amplification from primer pairs resulted in a
534-bp product for the V1a vasopressin receptor, a 391-bp
product for human OTR, and 208- and 266-bp products for human
ß-polymerase.
Determination of intracellular calcium levels in Hs578T
cells
Real-time recordings of [Ca2+]i were
performed on single cells, as previously described (20). In brief,
cells grown on glass coverslips for 48 h (unless otherwise stated)
were rinsed with physiological medium (KRH) composed of 125
mM NaCl, 5 mM KCl, 1.2 mM
KH2PO4, 1.2 mM MgSO4, 2
mM CaCl2, 6 mM glucose, and 25
mM HEPES-NaOH buffer, pH 7.4, and loaded with 2
µM fura-2/AM (Molecular Probes, Inc.,
Eugene, OR) for 50 min at 25 C to minimize dye compartmentalization.
The cells were then rinsed three times with KRH and incubated for 60
min at 4 C in the dark with KRH containing 0.1% BSA. Peptides were
prepared in the same solution, and 3 ml were delivered to the cells in
a Leiden dish after removal of the medium.
[Ca2+]i was determined by the method of
Grynkiewicz et al. (21), using 224 as the Kd for
fura-2 and Ca2+.
Determination of ERK-2 phosphorylation
Cells were maintained at confluence for 2 weeks on 35-mm
diameter dishes, and then kept in serum-free medium for 2 h. After
50-nM OT treatment with increasing time, the cells were
lysed, and ERK phosphorylation was analyzed by immunoblotting as
described previously, using antibody to phosphorylated and
nonphosphorylated ERK-2/1 (22). Blots were densitometrically scanned
and analyzed using a Dekmate III scanner and PDI IP software (PDI,
Hunting Station, NY). Quantification of pp42 (phosphorylated ERK-2) was
carried out by expressing its absorbance relative to the absorbance of
p42 (unphosphorylated ERK).
Determination of OTR mRNA levels using ribonuclease (RNase)
protection assay
RNA samples from Hs578T cells and myometrium were prepared
according to the method of Chomczynski and Sacchi (18). The RNA probe
used in the RNase protection studies was synthesized using RT-PCR, as
described under RT-PCR. The amplified 391-bp DNA was cloned into pCRII
(Invitrogen, Carlsbad, CA) and linearized with
HindIII, and labeled antisense transcripts were synthesized
by in vitro transcription with [-32P]UTP
(3000 Ci/mmol; New England Nuclear Corp., Boston, MA) and T7 RNA
polymerase, using a MAXIscript kit from Ambion, Inc.
(Austin TX). RNase protection assay was performed using a RPA II kit
(Ambion, Inc.) according to the manufacturer’s
instructions. Conditions that were optimized included using 20 and 5
µg total RNA from Hs578T cells and myometrium, respectively, and a
1:200 dilution of RNase A/T (200 U/ml RNase A and 10,000 U/ml RNase T).
Protected fragments were isolated on 5% acrylamide-8 M
urea gels, dried, and exposed to both x-ray film and a PhosphorImager
scanner screen (Molecular Dynamics, Inc., Sunnyvale, CA).
The intensity of the protected band was normalized to a glyceraldehyde
phosphate dehydrogenase (GAPDH) RNA-protected probe, which was
simultaneously analyzed in each sample. The GAPDH probe was obtained
from Ambion, Inc. and labeled using the same procedure as
that used for the OTR probe. RNA size markers were also obtained from
Ambion, Inc. and labeled according to the manufacturer’s
instructions. All experiments were performed in triplicate.
Measurement of glucocorticoid receptor (GR) levels in Hs578T cells
and demonstration of functional GR
Whole cell GR assays were performed using
[3H]triamcinolone acetonide (45 Ci/mmol; New England Nuclear Corp.) to detect high affinity binding to GR.
Briefly, confluent Hs578T cells were rinsed once with PBS and incubated
for 1 h at 37 C in the presence of 5 nM labeled
ligand. Nonspecific uptake was determined by coincubation with 1
µM unlabeled triamcinolone acetonide.
Functional GR was determined by transiently transfecting Hs578T cells with a plasmid containing a triplet glucocorticoid response element linked to a luciferase reporter gene (GRE/LUC). Dr. Allan Brasier (University of Texas Medical Branch, Galveston, TX) provided the GRE/LUC plasmid. Transient transfections were performed by calcium phosphate coprecipitation in triplicate 60-mm plates, using 10 µg GRE/LUC reporter vector, 0.5 µg RSVCAT (Rous sarcoma virus-cholamphenicol acetyltransferase) control for uniformity of transfection efficiency), and 9.5 µg carrier pGEM7Z plasmid DNA for each triplicate set of plates. Twenty-four hours after transfection, cells were rinsed, and fresh medium was added. Cells were cultured for an additional 20 h before adding 1 µM DEX for 4 h. Cells were rinsed three times with PBS and lysed with 0.3 ml detergent solution [25 mM Tris-phosphate (pH 7.8), 2 mM dithiothreitol, 2 mM 1,2-diaminocyclohexan-N,N,N,N-tetraacetic acid, 10% glycerol, and 1% Triton X-100]. Luciferase activity was determined using an AutoLumat luminometer (EG&G Wallac, Inc., Gaithersburg, MD), and the activity of each sample was normalized to CAT activity. CAT activity was determined using the enzyme-linked immunoassay kit and protocol described by Boehringer Mannheim (Indianapolis, IN).
PGE2 levels in Hs578T cells
Cells were maintained at confluence for 2 weeks in medium
containing 10% FBS. Four hours before stimulation with 50
nM OT, FBS was replaced with 0.1% (wt/vol) BSA in DMEM.
After cell treatment for 20 h, the medium was removed, and the
concentration of PGE2 was measured, using a
PGE2 enzyme immunoassay system from Amersham
(Aylesbury, UK). The sensitivity of the assay was 2.5 pg/ml, and the
intraassay coefficient of variation was 6.5%. The interassay
coefficient of variation was 6.7%.
Statistics
ANOVA followed by the Newman-Keuls test were used to determine
statistical differences between the means of treatment groups.
Differences at the P < 0.05 level were considered
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, lane 4), but
not the closely related V1a vasopressin receptor mRNA (Fig. 1, lane 5). Both OTR and V1a vasopressin receptor mRNA were
expressed in human myometrium (Fig. 1, lanes 2 and 3), in accordance
with ligand binding results (23). Binding studies were carried out
further, using [125I]OTA as the ligand. Scatchard
analysis showed a single class of high affinity binding site with an
apparent Kd value of 0.14 ± 0.083 (±SE)
nM (n = 3) and a binding capacity of 59 ± 22
(±SE) fmol/mg protein (n = 3; Fig. 2A). The specificity of binding was
determined by competition with several OT and vasopressin analogs. The
rank order of apparent Ki values was OTA > OT >
arginine vasopressin (AVP) = TGOT ([Thr4,
Gly7]oxytocin) > dDAVP
([deamino1, D-Arg8]AVP) >> OT
free acid (Fig. 2B). The results are consistent with the binding site
specificity of an OTR. OT had a lower apparent Ki than AVP.
TGOT, which has a lower oxytocic potency than OT in the
uterotonic assay, is more selective for the OTR than the
V1a receptor (24) and had a Ki value comparable
to that of AVP. dDAVP, which is more selective for
the V2 vasopressin receptor (25), had a relatively high
apparent Ki value. The results shown in Figs. 1 and 2
indicate that Hs578T cells express OTR, but not vasopressin
receptors.
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). The effects of the treatments were
additive; stimulation was 9-fold when DCC-FBS and DEX were combined
(Fig. 3A). The effects of DEX were probably mediated by GRs, as Hs578T
cells specifically took up 82 fmol [3H]triamcinolone
acetonide/µg DNA. The GRs were functional, as shown by DEX
stimulation of luciferase activity in Hs578T cells that were
transiently transfected with a GRE/LUC construct. Treatment of the
cells with 1 µM DEX for 4 h resulted in a 122
± 8 (±SE)-fold increase in luciferase activity (data not
shown). Cotreatment of cells with DCC-FBS/DEX and 1 µM
mifepristone reduced the level of increased OTR ligand binding activity
to that produced by DCC-FBS alone (data not shown). Pretreatment of
cells with actinomycin D (50 ng/ml) completely blocked the
up-regulation of OTR induced by DCC-FBS (10%) and/or 1
µM DEX (Fig. 3B). These results suggest that DCC-FBS and
DEX act by increasing OTR mRNA levels.
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, lane 9). Increased OTR mRNA
concentrations occurred as early as 2 h after treatment of
serum-starved cells with DCC-FBS/DEX and remained elevated through
24 h of treatment (Fig. 4A, lanes 2–7). Earlier time points were
not examined. Maximal OTR mRNA levels were found after 16 h of
treatment. Cotreatment of cells with actinomycin D completely inhibited
the DCC-FBS/DEX-induced rise in OTR mRNA levels at 16 h (Fig. 4A, lane 8). Similar increases in OTR mRNA occurred after treatments with
DCC-FBS or DEX alone, but at lower levels than the combined treatment
(Fig. 4B).
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).
Serum stimulation was lost upon heating FBS at 95 C for 10 min (Fig. 5B). The effects of specific inhibitors of signal pathways on serum
induction of OTR expression were examined to get a general idea of the
mechanisms of up-regulation. Pretreatment of serum-starved cells
with GF 109203X blocked DCC-FBS stimulated up-regulation of OTR in a
dose-dependent manner (Fig. 5B). The phospholipase C (PLC) inhibitor,
U73122 (1 µM), also inhibited the effects of DCC-FBS
(Fig. 5B). Inhibition of mitogen-activated protein kinase
kinase-1/2 activity with PD98059 resulted in about a 45%
increase in OTR ligand binding activity.
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). The EC50 was 1.9 ±
1.2 (SE) nM, and the maximal response was
obtained with 1 µM OT. The EC50 value is
comparable to the apparent Kd value for
[3H]OT binding by isolated rat mammary myoepithelial
cells (26). With increasing doses of OT, there was both an increase in
the fraction of cells responding and a decrease in the time lag between
administration of OT and initiation of the
[Ca2+]i transient (data not shown). Removal
of extracellular Ca2+ with EGTA had no apparent effect on
[Ca2+]i, suggesting that the increase in
[Ca2+]i was generated entirely from
intracellular stores (data not shown). Pretreatment of cells with OTA,
either 100 nM or 1 µM, completely inhibited
the calcium response to 10 nM OT (data not shown).
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), as would be expected with
down-regulation of OTRs. However, serum starvation had no effect on
bombesin (10 nM)-stimulated Ca2+ transients
(Fig. 7B). As the response to bombesin is mediated by a G
protein-coupled receptor, these results indicate that the effects of
serum starvation are not on G proteins in general, but are selective
for the OTR. Addition of 1 µM DEX and/or DCC-FBS (10%)
for 24 h resulted in restoration of the Ca2+ response
to increasing concentrations of OT (Fig. 7, C–E), consistent with the
up-regulation of OTRs. The effects of DEX were blocked by incubation of
cells with a 10-fold excess of the glucocorticoid antagonist,
mifepristone (Fig. 7F). These findings indicate that the down- and
up-regulation of OTR concentrations with DCC-FBS and DEX dictate the
Ca2+ response of Hs578T cells to OT.
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, lanes 1–7). Phosphorylation was
first apparent about 5 min after OT treatment, reached maximal levels
at about 10 min, and was sustained for up to 1 h (Fig. 8A).
Pretreatment of Hs578T cells with OTA (1 µM) for 15 min
completely blocked the stimulation by 50 nM OT at 10 min
after addition of the agonist (Fig. 7A
, lane 8). Pretreatment of cells
with the MEK1/2 inhibitor PD98059 (1 µM) also completely
blocked OT-stimulated ERK-2 phosphorylation (Fig. 8B). Pretreating
cells with 10 µM GF109203X, a selective PKC inhibitor,
blocked OT-induced ERK-2 phosphorylation (Fig. 8C), indicating that the
phosphorylation of MAP kinase kinase kinase (Raf-1) is mediated by PKC,
as shown previously in CHO cells transfected with the OTR (22).
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).
This increase was inhibited by preincubation with the PKC and MEK1/2
inhibitors, GF 109203X and PD98059, respectively (Fig. 8D). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mechanisms of up-regulation of OTR in Hs578T cells
Hs578T cells contain functional GR, as shown by specific
[3H]triamcinolone acetonide uptake and by DEX stimulation
of luciferase activity in cells that were transiently transfected with
a GRE/LUC reporter construct. DEX treatment of Hs578T cells resulted in
about a 4-fold up-regulation of OTRs. Increasing amounts of FBS led to
higher OTR concentrations until a maximal response was reached with
60% FBS. The exposure of human myometrial cells in culture to
FBS might also explain why the concentration of
OTRs rises spontaneously over time (35). The factors in FBS that are
responsible for the up-regulation of OTR are not currently known. Heat
treatment (95 C) of FBS caused a loss in its ability to up-regulate
OTRs in Hs578T cells, suggesting that proteins impart stimulation.
Because DEX was stimulatory, FBS was stripped of endogenous steroids by
treatment with DCC. The effects of FBS, however, were independent of
glucocorticoids, as coincubation with the GR antagonist, mifepristone
(1 µM), did not reduce the effects of DCC-FBS (data not
shown). The effects of DCC-FBS on OTR ligand binding levels appear to
be mediated by G protein-coupled receptors, as inhibitors of both PLC
and PKC activities were effective in blocking the serum-induced rise in
OTR concentrations.
Up-regulation of OTR in Hs578T cells occurred at the mRNA level. Inhibition of transcription with actinomycin D blocked both FBS- and DEX-induced rises in ligand binding activity. As was the case with ligand binding activity, the effects of FBS and DEX on OTR mRNA levels were additive. Increased OTR mRNA levels were observed after 2 h, the earliest time point studied, and maximal levels were reached at 16 h. The results suggest that both FBS and DEX act at the genomic level to induce OTR mRNA transcription. Glucocorticoids have also been shown to up-regulate OTRs in rabbit amnion cells at the transcriptional level (11).
Hs578T cells are the first established cell line in which OTR regulation by glucocorticoid and serum factor(s) has been demonstrated. Bale and Dorsa showed that estrogen treatment of MCF7 cells resulted in a 17-fold increase in [125I]OTA binding (36). More recently, these workers showed that TPA and forskolin increased [125I]OTA binding by MCF7 cells by 40- and 10-fold, respectively (37). As we have shown in the present studies, PKC plays a pivotal role in the up-regulation of OTR concentrations. Because the up-regulation of OTR-binding sites is associated with increases in OTR mRNA levels, PKC appears to mediate increased transcription of the OTR gene. Indeed, we have shown that c-Fos and c-Jun play an important role in regulating OTR gene expression in Hs578T cells (19). Activation of OTR transcription is more complex, however, because the effects of DCC-FBS or DEX cannot be mimicked by TPA stimulation of PKC activity alone (data not shown). Our recent findings show that a critical guanine adenine binding protein-binding site in the human OTR promoter is required for basal, serum, and c-Fos/c-Jun induction of OTR gene expression (19).
OT signal pathways in Hs578T cells
OT stimulated [Ca2+]i transients in
Hs578T cells that were both dose and time dependent. Ca2+
responsiveness to OT varied commensurately with down- and up-regulation
of OTRs. OTR concentrations were down-regulated by serum starvation
under conditions where there did not appear to be any impaired ability
of the cells to respond to bombesin with an increase in
[Ca2+]i. Bombesin binds to its cognate
gastrin-releasing peptide receptor on the cell membrane, resulting in
an increase in [Ca2+]i through a
Gq protein-coupled response (38).
Treatment of Hs578T cells with OT resulted in the phosphorylation of ERK-2. A PKC pathway, as evidenced by inhibition by GF 109203X, mediates this process. Depending on the cell type, ERK activation results in either proliferation or differentiation (39). The role of OT-induced ERK-2 phosphorylation in Hs578T cells is not completely understood at the present time, as it does not appear to involve any significant short term effects on growth that we could observe. We found, however, that the MEK1/2 inhibitor (PD98059), which blocked OT-stimulated ERK-2 phosphorylation, almost completely eliminated both basal and OT-stimulated PGE2 synthesis. Hs578T cells growing in log phase appear to lack a source of endogenous arachidonic acid as a precursor for PGE2 synthesis (28). These cells have a functional cyclooxygenase system, as provision of arachidonic acid allowed a sharp increase in the basal synthesis of PGE2. When the cells were maintained confluent for 2 weeks, OT stimulated a 2-fold increase in PGE2 production by 24 h, indicating that the cells had acquired sufficient phospholipids to serve as a source of endogenous arachidonic acid. It would appear, therefore, that OT acts primarily at the level of making arachidonic acid available for PGE2 synthesis.
The importance of OTR in breast tumor cells
Among 57 breast cancer patients studied, Ito et al.
(13) detected OTR immunoreactivity in 52 (91.2%) by
immunohistochemistry using a monoclonal antibody. The expression of the
OTR in positively stained samples was confirmed by means of Northern
blotting and RT-PCR. Bussolati et al. (12) showed that OTRs
in human breast were detected in intraductal cells in benign
hyperplastic lesions. OTRs were also demonstrated in cases of primary
and metastatic carcinomas of the breast. It has been suggested that the
interaction between OT and OTR might play a role in the origin and
evolution of nonneoplastic lesions and carcinomas of the breast (12).
OTRs also have been found in four breast cancer cell lines (MCF7,
MDA-MB-231, MDA-MB-361, and MDA-MB-468), using monoclonal antibody to
the OTR and flow cytometry (13). However, OT had no significant effect
on cell growth during 7 days of culture (13). These findings are
controversial, as other workers have found that OT inhibited the growth
of MCF7, T47D, and MDA-MB231 human breast cancer cell lines (14). OT
has also been found to inhibit cell proliferation and tumor growth of
xenografts of mouse mammary and colon carcinomas (TS/A and C26 tumors)
and of a rat mammary carcinoma (40). Yet other studies have shown that
OT stimulated the growth of MCF7 cells (30).
Detection of OTR in breast tissue or tumor cells grown in culture by ligand binding or immunohistochemical or immunocytochemical assays might not be relevant to the effects of OT if the receptors were not functional. The present studies clearly demonstrate that OTR in Hs578T cells are functionally coupled. Specifically, we have shown that OT stimulates Ca2+ signaling, ERK-2 phosphorylation, and PGE2 synthesis. As we showed in the Ca2+ transient studies, the up-regulation of OTRs dictates the responsiveness of cells to OT, as is the case in other OT target tissues (1).
In summary, we have demonstrated the presence of functional OTRs in breast tumor cells that are not of myoepithelial origin. The signal pathways emanating from occupancy of OTR-binding sites are the same as those stimulated by OT in other cell types and are reflective of the OTR being coupled to G proteins. OTRs are up-regulated by FBS and DEX, and up-regulation by FBS is a PKC-mediated process. Hs578T cells have already proven useful in studies on expression of transfected OTR promoter-reporter constructs under conditions where treatment of the cells with FBS resulted in increased reporter activity (19). The identity of factors in serum involved in induction of OTR gene expression remains to be determined. The mechanisms of down-regulation of OTR can be elucidated in future studies by examining degradation of OTR protein in Hs578T cells through possible protease-mediated events and/or changes in transcriptional or posttranscriptional regulation of the OTR gene. Hs578T cells also provide a useful paradigm for studying additional signal pathways elicited by OT. Finally, Hs578T cells might be useful in studies of the role of OT in breast cancer.
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Acknowledgments |
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Footnotes |
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Received October 23, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/ß binding site involved in
the induction of oxytocin receptor gene expression in human breast
cells. Potentiation by c-Fos/c-Jun. Endocrinology 140:2268–2279
downregulates expression of the oxytocin receptor in cultured human
myometrial cells. Am J Physiol 271:E840—E846
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S. Hoare, J. A. Copland, T. G. Wood, Y.-J. Jeng, M. G. Izban, and M. S. Soloff Identification of a GABP{alpha}/{beta} Binding Site Involved in the Induction of Oxytocin Receptor Gene Expression in Human Breast Cells. Potentiation by c-Fos/c-Jun Endocrinology, May 1, 1999; 140(5): 2268 - 2279. [Abstract] [Full Text]
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